Bottom Line:
Cells overexpressing Staufen1 differentiated poorly, as evidenced by reductions in the differentiation and fusion indices and decreases in MyoD, myogenin, MEF2A, and MEF2C, independently of Staufen-mediated mRNA decay.By contrast, the knockdown of Staufen1 decreased c-myc levels in myoblasts.Collectively our results show that Staufen1 is highly expressed during early stages of differentiation/development and that it can impair differentiation by regulating c-myc, thereby highlighting the multifunctional role of Staufen1 in skeletal muscle cells.

Mentions:
We then sought to investigate whether Staufen1 regulates expression of the myogenic regulatory factor MyoD, a crucial regulator of myogenesis (Tapscott et al., 1988; Berkes and Tapscott, 2005; Buckingham and Vincent, 2009; Bismuth and Relaix, 2010; Bentzinger et al., 2012). We found that during proliferation and differentiation, expression of MyoD was significantly reduced (p < 0.05) in both Staufen1-HA–overexpressing clones compared with control cells (Figure 6, A–C). To assess whether the addition of exogenous MyoD could rescue the differentiation defect, we performed MyoD add-back experiments. Briefly, stable cell lines were transfected with MyoD-Flag-myc or control vectors. After transfection, cells were allowed to differentiate for 3 d. First, the increase in MyoD expression was confirmed by Western blot using MyoD antibodies. In these blots, the lower band corresponds to endogenous MyoD and the upper band to ectopic MyoD-Flag-myc (Figure 6D). Note that we were able to restore normal MyoD levels in Staufen1-HA stable cell lines (compare CTL+pcDNA3 and #25+MyoD). Then we assessed the efficiency of differentiation by measuring expression of myogenin and by immunofluorescence using a pan-MyHC antibody. As illustrated in Figure 6, E–G, no rescue of differentiation was observed in the Staufen1-HA stable cell line upon addition of MyoD-Flag-myc (compare both Staufen1-HA cells transfected with MyoD-Flag-myc to control cells transfected with MyoD-Flag-myc and to Staufen1-HA cells without MyoD-Flag-myc). Taken together, these findings show that the inhibition of differentiation induced by Staufen1 is MyoD independent.

Mentions:
We then sought to investigate whether Staufen1 regulates expression of the myogenic regulatory factor MyoD, a crucial regulator of myogenesis (Tapscott et al., 1988; Berkes and Tapscott, 2005; Buckingham and Vincent, 2009; Bismuth and Relaix, 2010; Bentzinger et al., 2012). We found that during proliferation and differentiation, expression of MyoD was significantly reduced (p < 0.05) in both Staufen1-HA–overexpressing clones compared with control cells (Figure 6, A–C). To assess whether the addition of exogenous MyoD could rescue the differentiation defect, we performed MyoD add-back experiments. Briefly, stable cell lines were transfected with MyoD-Flag-myc or control vectors. After transfection, cells were allowed to differentiate for 3 d. First, the increase in MyoD expression was confirmed by Western blot using MyoD antibodies. In these blots, the lower band corresponds to endogenous MyoD and the upper band to ectopic MyoD-Flag-myc (Figure 6D). Note that we were able to restore normal MyoD levels in Staufen1-HA stable cell lines (compare CTL+pcDNA3 and #25+MyoD). Then we assessed the efficiency of differentiation by measuring expression of myogenin and by immunofluorescence using a pan-MyHC antibody. As illustrated in Figure 6, E–G, no rescue of differentiation was observed in the Staufen1-HA stable cell line upon addition of MyoD-Flag-myc (compare both Staufen1-HA cells transfected with MyoD-Flag-myc to control cells transfected with MyoD-Flag-myc and to Staufen1-HA cells without MyoD-Flag-myc). Taken together, these findings show that the inhibition of differentiation induced by Staufen1 is MyoD independent.

Bottom Line:
Cells overexpressing Staufen1 differentiated poorly, as evidenced by reductions in the differentiation and fusion indices and decreases in MyoD, myogenin, MEF2A, and MEF2C, independently of Staufen-mediated mRNA decay.By contrast, the knockdown of Staufen1 decreased c-myc levels in myoblasts.Collectively our results show that Staufen1 is highly expressed during early stages of differentiation/development and that it can impair differentiation by regulating c-myc, thereby highlighting the multifunctional role of Staufen1 in skeletal muscle cells.